Background. It is well established that the energy source of muscle contraction is derived from ATP hydrolysis by actin and myosin. Our long term goal is to elucidate the mechanism of the energy transduction from ATP hydrolysis into muscle contraction. It has been expected for some time that the transduction involve some structural changes at the molecular level. As myosin (acting as an enzyme) hydrolyzes ATP, it passes through several intermediate states including the binding of the nucleotide, the hydrolysis step, and the release of the hydrolysis products. It is thought that force is generated (and hence movement) during the release of the hydrolysis products. The atomic structures of the myosin head (S1) have revealed ligand-dependent differences, which may very well correspond to changes induced by ATP hydrolysis in the actomyosin complex. However, crystal structures of isolated S1 and an actin monomer alone do not necessarily reveal the in vivo structural changes that lead to force generation. To gain full understanding of the contraction mechanism, it is critical to determine the in vivo structures in a fully functioning muscle cell. In particular, if the actomyosin affinity is weak, a noninvasive technique would be necessary for preserving the structures. It has also been recognized that in order to generate force, some strain (hence some structural distortion) must be sustained by the links formed by myosins (the crossbridges) between the filaments. Due to the mismatch between the periodicities of the filaments, there is a distribution of strains sustained by the links. Therefore, a technique that can provide information about the distributions and orientations of the crossbridges in the filament lattice during ATP hydrolysis cycle is critical. X-ray diffraction from permeabilized muscle cells, the technique used in the present study, is one of the few techniques that reveal the structures as they occur in muscle cells, albeit at relatively low resolution. A wealth of structural information has been obtained. In FY99, we have identified that those myosin heads arranged in an ordered helical array on the surface of the myosin filament are in the M.ADP.Pi state where the ATP has been cleaved but the products not yet released. Since then, we have concentrated our efforts in elucidating the structures of the low affinity states (A.M.ATP and A.M.ADP.Pi where myosins with bound ATP or hydrolyzed products are attached to actin). The myosin must first bind to actin in the low affinity states before going onto force generation. In FY99 the major characteristics of the state of A.M.ATP were determined, i.e. the attachment with multiple orientations and location of binding site on actin. Objectives. Our strategy over the past few years has been to characterize the structures of the individual hydrolysis intermediate states, such that structural transitions involved in force generation might be understood. To achieve this, we have used ATP analogues and chemically modified the myosins to "trap" the muscle in one of the intermediate states. To shorten the exposure time as much as possible so as to maintain viability of the muscle tissue, intense synchrotron radiation sources such as those at the Brookhaven National Laboratory have been routinely used as the X-ray source. Results. In FY00, we further refine the structures of the relaxed muscle in the low affinity states (A.M.ATP and A.M.ADP.Pi). One surprising new result is that the unattached head of myosin (there are two heads to each myosin) appears to be located away from the actin filament. The whereabouts of the unattached head have eluded researcher for many years. One may begin to ask questions e.g. cooperativity between the two heads under in vivo conditions. In FY00 continuing our efforts in identifying structures of the ATP hydrolysis, we have obtained preliminary data on the A.M.ADP.Pi state. Although the interpretation of the results is not yet complete, it is clear that the binding characteristics are different from that of the pre-cleavage state A.M.ATP. These findings provide a significant step towards delineating the structure-function relationship of muscle. During FY00, we probed further into the cause of the conformational change in myosin. As it was suggested by us (FY99) that the helical order of the rabbit skeletal myosin filaments at temperatures >20 ?C was a consequence of the dependence on temperature of the hydrolytic step of myosin ATPase and the requirement that hydrolysis products (eg ADP.Pi) be bound at the active site. An alternative hypothesis is that temperature directly affects the conformation of myosin and that myosin heads need to be in a particular conformation for helical order in the filament. To discriminate between these two possibilities, we have studied the effect on the helical order of myosin heads in rabbit psoas muscle in the presence of non-hydrolyzable ligands. We show that in the presence of ADP + vanadate, which mimics the transition state between ATP and hydrolysis products, myosin filaments are helically ordered at high temperatures but still display a marked dependence on temperature. In the presence of AMPPNP, a substrate analog, the myosin filaments are partially ordered at 35 ?C and again there is a dependence of order on temperature. Other analogs such as ADP+BeFx and ATPgS showed similar temperature dependence. We conclude that helical order reflects the proportion of myosin heads in a conformation that corresponds to the atomic "closed" form and it is affected by temperature as well as by ligands. We suggest that the temperature dependence of the hydrolytic step of myosin ATPase is attributable to the effect of temperature on myosin conformation. Conclusions. With the conclusion of studying the A.M.ATP state, distinct actin and myosin filament structures associated with seven out of eight major biochemical states have been identified. With the studies of the A.M.ADP.Pi state expected to be completed during FY01 or shortly afterwards, a picture of the process of force generation associated with ATP hydrolysis could emerge. These findings will provide a significant step towards delineating the structure-function relationship of muscle.